LMNA Co-Regulated Gene Expression as a Suitable Readout after Precise Gene Correction

Area of Science:

• Molecular Biology
• Genetics
• Cell Biology

Background:

• LMNA-related muscular dystrophy is a progressive disorder caused by mutations in the LMNA gene.
• Lamin A/C proteins, encoded by LMNA, are crucial nuclear envelope components but absent in pluripotent stem cells.
• Direct loss of function of lamin A/C is not typically observed in patient-derived cells, posing challenges for evaluating gene editing outcomes.

Purpose of the Study:

• To develop a method for evaluating functional recovery of LMNA after gene editing in LMNA-related muscular dystrophy.
• To induce lamin A/C expression in patient-derived induced pluripotent stem cells (iPSCs) for functional analysis.
• To demonstrate the efficacy of CRISPR/Cas9-based gene correction for LMNA mutations.

Main Methods:

• Induced lamin A/C expression in iPSCs from patients with specific LMNA mutations (c.1366A > G and c.1494G > T) using a three-day serum-induced differentiation protocol.
• Analyzed expression profiles of co-regulated genes, including COL1A2 and S100A6.
• Performed precise gene editing of the LMNA c.1366A > G mutation using a near-PAMless cytosine base editor.

Main Results:

• Achieved 100% efficiency in repairing the LMNA c.1366A > G mutation in individual iPSC clones.
• The differentiation protocol provided a functional readout, showing increased lamin A/C expression.
• Demonstrated normalization of co-regulated gene expression (e.g., COL1A2, S100A6) following gene correction.

Conclusions:

• CRISPR/Cas9-mediated gene correction is a powerful therapeutic strategy for LMNA-related muscular dystrophy.
• The developed fast differentiation protocol serves as an effective outcome measure for assessing gene editing success.
• These findings offer new hope for therapies in a disease with previously limited treatment perspectives.